The Ubiquitous Nature of Multivesicular Release

Feature Review

The ubiquitous nature of multivesicular release

1 1 2

Stephanie Rudolph , Ming-Chi Tsai , Henrique von Gersdorff , and

Jacques I. Wadiche

Department of Neurobiology and Evelyn McKnight Brain Institute, University of Alabama at Birmingham, Birmingham,

AL 35294, USA

The Vollum Institute, Oregon Health and Science University, Portland, OR 97239, USA

‘Simplicity is prerequisite for reliability’ (E.W. Dijkstra [1]) ﬂuctuations of evoked synaptic responses [11] and high

Presynaptic action potentials trigger the fusion of vesicles transmitter concentration in the synaptic cleft [12]. To

to release neurotransmitter onto postsynaptic neurons. reconcile these ﬁndings, a more ﬂexible hypothesis pro-

Each release site was originally thought to liberate at poses that multiple vesicles can be released per active zone

most one vesicle per action potential in a probabilistic with each action potential, deﬁned as MVR. Indeed, stud-

fashion, rendering synaptic transmission unreliable. How- ies have established that MVR occurs at many inhibitory

ever, the simultaneous release of several vesicles, or and excitatory synapses throughout the brain, including

multivesicular release (MVR), represents a simple mecha-

nism to overcome the intrinsic unreliability of synaptic

transmission. MVR was initially identiﬁed at specialized

synapses but is now known to be common throughout Glossary

the brain. MVR determines the temporal and spatial dis-

Active zone: ultrastructurally and functionally specialized area at the pre-

persion of transmitter, controls the extent of receptor synaptic terminal where readily-releasable vesicles fuse (see also Release site).

Coefficient of variation (CV): standard deviation s divided by the mean (e.g., s

activation, and contributes to adapting synaptic strength

of current amplitude fluctuations and mean current). CV is a normalized

during plasticity and neuromodulation. MVR consequent- measure of relative variability within a distribution (e.g., numerous trials of an

ly represents a widespread mechanism that extends the evoked synaptic current).

Depletion: temporal unavailability of releasable vesicles as a result of foregoing

dynamic range of synaptic processing.

release. Vesicular depletion can cause a decrease in synaptic strength.

Desensitization: receptors bound by transmitter but in a non-conducting state

MVR occurs throughout the brain can contribute to failure of receptor opening following repeated or prolonged

exposure to neurotransmitter. Desensitization can cause a decrease in synaptic

Fast chemical communication between neurons occurs at

strength.

ultrastructurally deﬁned synaptic junctions through the Desynchronization: the temporal jitter of vesicle fusion between synaptic

release of neurotransmitters. At each presynaptic release release sites (intersite asynchrony) or within a release site (intrasite

asynchrony).

site, neurotransmitter-ﬁlled vesicles are docked on the

EPSC/EPSP: excitatory postsynaptic current/potential.

plasma membrane ready to fuse upon the arrival of an Fast-off/low-affinity antagonist: rapidly-dissociating competitive antagonist.

IPSC/IPSP:

action potential (Figure 1Ai,Bi). Vesicle fusion and neuro- inhibitory postsynaptic current/potential.

Multivesicular release (MVR): the release of >1 vesicles per active zone in

transmitter release then result in receptor activation. The

response to a single presynaptic action potential. Transmitter released by

strength of the synaptic signal at the postsynaptic mem- multiple vesicles interacts with a common population of postsynaptic

receptors.

brane is determined by the number of release sites (N; see

Quantal theory: postulates that the spontaneously occurring miniature currents/

Glossary), the probability that a vesicle is released (Pr),

potentials represent the basic element of the evoked synaptic current/potential.

and the amplitude of the postsynaptic response elicited by Receptor occupancy: the percentage of receptors bound by released transmit-

ter. 100% occupancy refers to receptor saturation.

the content of each synaptic vesicle (q) [2]. Seminal work

Release site: site within the active zone where a vesicle undergoes fusion. It

correlating morphology with physiology led to the idea that

remains controversial whether release sites are clearly defined molecularly or

an action potential allows the stochastic fusion of, at most, whether release can take place at any location in the active zone. For the

purpose of this review, we define the active zone as a structure that provides

one vesicle at each release site (univesicular release; UVR)

release sites, and a single active zone can have multiple release sites.

[3–8]. Under this model the maximum number of vesicles

Slow-off/high-affinity antagonist: slowly-dissociating competitive antagonist.

released corresponds to the number of anatomically de- Spillover: refers to transmitter escaping the synaptic cleft that activates

extrasynaptic receptors located on the releasing cell or receptors on adjacent

ﬁned release sites, N [9]. The tenet of UVR was thought to

cells.

apply to most synapses (see [10] for review); however, Synaptic strength: defines the average response amplitude at a synaptic

several observations are inconsistent with the ‘one site, contact in response to an action potential. The total number of release sites,

readily releasable vesicles, Pr, and postsynaptic factors such as single receptor

one vesicle’ hypothesis, including considerable amplitude

conductance and receptor density, determine synaptic strength.

Univesicular release (UVR): the release of 1 vesicle per active zone in

response to a single presynaptic action potential.

Corresponding authors: Rudolph, S. ([email protected]);

Variance-mean analysis (VMA): a statistical method to estimate synaptic

Wadiche, J.I. ([email protected]).

quantal parameters N, Pr, and q.

0166-2236/ Vesicular release probability (Pr): the likelihood that a vesicle fuses upon arrival

of an action potential at any release site within an active zone.

428 Trends in Neurosciences, July 2015, Vol. 38, No. 7

Feature Review Trends in Neurosciences July 2015, Vol. 38, No. 7

(Ai) (Bi)

(Aii) (Bii)

(C)

UVR MVR dMVR

[transmier] 4 mM 10 ms

1 mM 5 mM 2 mM EPSC 200 µs

500 pA 10 ms

TRENDS in Neurosciences

Figure 1. Multiquantal release at single synapses. (Ai,ii) Electron micrograph and corresponding cartoon of a hippocampal synapse imaged following rapid freezing.

Arrows indicate the presence of three docked vesicles at the active zone. (Bi) Electron micrograph of two synaptic vesicles fusing with the within an active zone

simultaneously. (Bii) Cartoon representation of (Bi) illustrating neurotransmitter released from MVR interacts with a common pool of postsynaptic receptors (green and

orange). Adapted from [22]. (C) Overview of different release modalities and their consequences for postsynaptic currents. UVR causes a low-concentration, short-lived

transmitter transient in the synaptic cleft that results in a small EPSC (left). Synchronous fusion of several vesicles, MVR, results in elevated synaptic transmitter

concentration and a large EPSC (middle). Desynchronized MVR prolongs but reduces the transmitter concentration transient, resulting in a smaller and slowed EPSC (right).

Adapted from [39]. Abbreviations: EPSC, excitatory postsynaptic current; MVR, multivesicular release; UVR, univesicular release.

hippocampus, cerebral cortex, cerebellum, hypothalamus, underlining MVR as an indispensable mechanism for neu-

and at sensory synapses [12–21]. Due to the sub-millisecond ronal computation.

speed of vesicle fusion direct electron microscopic evidence of

multiple vesicle fusions (omega ﬁgures) at single release How MVR shapes synaptic transmission

sites has been rare (but see [22,23]) (Figure 1). Nevertheless, Receptor occupancy and transmitter concentration

functional data from many studies strongly suggests that Heterogeneity in synapse size, vesicular transmitter con-

MVR is a widespread phenomenon among synapses – more tent, transmitter clearance, and receptor afﬁnity contrib-

prevalent than originally assumed. ute to the variability of neurotransmitter-receptor

In this review we delineate the evidence and conse- occupancy levels [12,26–30]. MVR can only increase

quences of MVR, including its effects on the time course strength at individual synapses when receptor occupancy

and concentration of transmitter, receptor occupancy, and by transmitter released from a single vesicle is sufﬁciently

desensitization. We will examine the experimental low. Indeed, evidence from many synapses suggests that

approaches that have helped to identify MVR, explore transmitter from one vesicle is unlikely to fully occupy

how MVR shapes synapse properties, and argue that postsynaptic receptors [11,31,32]. For example, at excit-

MVR not only promotes synaptic strength and reliability atory hippocampal synapses, transmitter from multiple

but also enhances the dynamic range of a neuron during vesicles can interact with a common population of postsyn-

plasticity and neuromodulation [24,25]. Finally, we review aptic receptors to increase synaptic strength (Figure 1B),

the role of MVR in a wide range of physiological contexts, and Pr determines the extent of receptor occupancy

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Feature Review Trends in Neurosciences July 2015, Vol. 38, No. 7

[12]. These observations are consistent with several stud- distinction when release sites are sufﬁciently far apart.

ies showing increased receptor occupancy under high Pr At many synapses, glial cell processes that enwrap synap-

conditions [33–35]. Low receptor occupancy is particularly ses establish an anatomical diffusion barrier and provide

evident between cholecystokinin (CCK)-positive interneur- high densities of excitatory amino acid transporters

ons and CA3 pyramidal cells where MVR enhances the (EAATs) to separate release sites. Together with perisy-

inhibitory postsynaptic current (IPSC) by several fold naptic neuronal EAATs, these transporters rapidly buffer

[30]. Hence, MVR allows synapses with low receptor occu- and uptake glutamate to spatially and temporally conﬁne

pancy to enhance their dynamic range. However, the ex- transmitter spread [55] and maintain extracellular gluta-

tent of receptor occupancy varies markedly across mate concentrations in the nM range [56].

synapses. For example, the effect of MVR on IPSC ampli- In accord with experimental ﬁndings that only high levels

tude is minimal at synapses between cerebellar molecular of MVR can temporarily overwhelm glutamate clearance

interneurons (MLIs), implying near full occupancy of post- mechanisms [46,57], and that spillover is more apparent at

synaptic receptors by the contents of a single vesicle synapses lacking glial ensheathment [58], simulations

[13]. While such high-occupancy synapses forfeit dynamic suggest that synapse crosstalk occurs only when near-

range due to insensitivity to changes in Pr, they operate synchronous MVR is combined with a lack of astrocytic

with greater ﬁdelity. Together, these examples demon- coverage [40,59]. Hence, although MVR signiﬁcantly

strate that the degree of receptor occupancy is an impor- increases transmitter concentration, efﬁcient clearance

tant consideration in assessing the consequences of MVR. largely eliminates spillover, highlighting that independence

of glutamatergic synapses remains preserved even during

The temporal and spatial spread of transmitter MVR. As a result, at excitatory synapses with low MVR such

Evoked transmission across synapses is typically tightly as Schaffer collateral (SC)–CA1 contacts, spillover is most

timed to the presynaptic stimulation [36], suggesting tem- evident in response to repetitive stimulation and when

poral coordination across release sites (Figure 1C, left). monitored with high-afﬁnity, slowly desensitizing NMDARs

Nevertheless, MVR has been reported to be either highly that are more suitable to detect lower glutamate concentra-

coordinated [16,37] (Figure 1C, center) or desynchronous tions [44,60]. However, the near-simultaneous stimulation

[38,39] in a manner that alters the neurotransmitter of adjacent ﬁbers that occurs with many experimental para-

concentration at postsynaptic receptors (Figure 1C). Numer- digms may exaggerate the consequences of spillover

ical simulations suggest that desynchronization of MVR can [44,61]. A notable exception is glutamate spillover from

differentially inﬂuence glutamatergic AMPA receptor the climbing ﬁber (CF) to MLIs in the cerebellar cortex.

(AMPAR) and NMDA receptor (NMDAR) activation Here, high MVR allows glutamate to activate interneuron

[40]. Near-synchronous MVR (tens of microseconds) increases AMPARs and NMDARs, generating slow and long-lasting

AMPAR activation supra-linearly, while more pronounced excitation [62] that increases ﬁring for tens of milliseconds

temporal dispersion of MVR (several milliseconds) can result (see below and [63,64]).

in AMPAR desensitization. Conversely, vesicle release syn- Another potential consequence of prolonged transmitter

chrony has little effect on NMDAR activation. Therefore, presence following MVR is receptor desensitization. De-

activity-dependent desynchronization of MVR might not only sensitization can diminish receptor availability, contribute

alter the time course of the synaptic currents but also its to short-term depression, shape the time course of synaptic

relative receptor contributions. Although the molecular mech- currents, and is most common among synapses with clus-

anisms underlying MVR are currently unknown (Boxes 2 and tered release sites, high Pr, and slowed transmitter clear-

3), it is tempting to speculate how it regulates both synaptic ance [65–68]. Hence, desensitization occurs at some but not

integration and the recruitment of downstream signaling. all MVR synapses. For example, there is minimal desensi-

The elevated transmitter concentrations that result tization of AMPARs at synapses where glutamate is rap-

from MVR can, under some circumstances when transmit- idly cleared by glial and neuronal EAATs (CF–Purkinje

ter clearance is impeded by restricted geometry, promote cell (CF–PC) synapses) [69,70], whereas MVR results in

rebinding of transmitter to synaptic receptors [41]. MVR profound desensitization at retinogeniculate contacts

can also promote transmitter escape from the synaptic cleft [51]. In conclusion, MVR can exacerbate desensitization,

to activate extrasynaptic receptors [17,42–46]. Both re- but synapse tortuosity, glial coverage, receptor composi-

binding and spillover are expected to prolong the duration tion, and the spacing of release sites are likely the main

of synaptic currents, and indeed there is a correlation factors [71,72]. Less is known about how MVR of GABA

between MVR and increased decay time [30,47]. Transmit- affects spillover and desensitization. However, the lack of

ter spillover most likely occurs when the transmitter con- efﬁcient clearance mechanisms predicts a greater extent of

centration is high, for example after MVR, when release GABA escape from the synaptic cleft, perhaps with more

sites are closely spaced, or in the absence of glial diffusion pronounced effects on GABAA receptor desensitization and

barriers and uptake machinery [48–51]. A clear distinction tonic inhibitory currents [73–75].

between spillover from a neighboring release site and MVR

is often difﬁcult because both phenomena can result in Vesicle release probability as a predictor of MVR?

prolonged presence of transmitter in the synaptic cleft and What is the principal determinant of MVR? The most

at extrasynaptic sites [52–54]. However, studies that use parsimonious hypothesis is that Pr regulates whether

low-afﬁnity antagonists suggest that spillover does not multiple vesicles are released concurrently. If each docked

increase peak transmitter concentration in the cleft, vesicle can fuse independently in response to an action

whereas MVR does [14,17], allowing pharmacological potential, then the simultaneous release of multiple vesicles

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Feature Review Trends in Neurosciences July 2015, Vol. 38, No. 7

will occur more readily at synapses with higher Pr. For has been proposed as an alternative mechanism to explain

example, if two docked vesicles are each released with a multiphasic EPSCs [90].

probability of 0.9, then the simultaneous release of two MVR is prominent throughout the retinal circuit. Rod

vesicles will occur with a probability of 0.81. Consistent bipolar cells (RBCs) receive input from light-sensing rods

with this idea, the exceptionally high Pr at the CF terminal and relay the visual information to AII amacrine cells. This

(0.35–0.9) [76,77] coincides with signiﬁcant MVR. Yet, retinal ribbon synapse exhibits highly synchronous MVR

some high Pr synapses display exclusively UVR [6,7], where- [16] that is modulated by the number of open calcium

as various low Pr synapses exhibit MVR [15,17,34,78], dem- channels. MVR requires at least two open calcium channels

onstrating that Pr does not always reliably predict the to trigger the release of multiple vesicles from one ribbon

incidence of MVR. release site [98]. Astoundingly, transmitter from the 2–4

However, increasing Pr promotes MVR. Many low Pr simultaneously released vesicles onto amacrine cells neither

synapses generate small UVR postsynaptic currents fully occupies nor desensitizes postsynaptic AMPARs, allow-

(PSCs) that are prone to failure, but repetitive stimulation ing linear summation of each vesicle and a wide range of

can facilitate PSC amplitude. Release models predict that synaptic operation [93,99]. By contrast, MVR at cone bipolar

this facilitation depends on an increase in the number of cell–ganglion cell synapses fully occupies AMPARs and

fusion-ready vesicles and/or Pr [79]. Activity-dependent activates perisynaptically located NMDARs in a concentra-

long- and short-term increases of MVR, for example during tion-dependent manner [45,100,101]. In addition, at some

facilitation, augmentation, and long-term potentiation bipolar cell terminals presynaptic activation of GABAC

(LTP), have been observed at numerous synapses with receptors regulates Pr and MVR, and determines the extent

low initial Pr [11,17,34,80,81], supporting the view that of spillover and NMDAR activation [100,102,103].

high Pr promotes MVR. Considering inter-terminal het- In summary, sensory synapses take advantage of MVR

erogeneity in synapse size, Pr, number of docked vesicles, to regulate ﬁring duration and frequency, and also to

and molecular make-up [82–84], it is also probable that the ensure reliable and sustained postsynaptic ﬁring at high

extent of MVR differs strongly from terminal to terminal frequencies. Moreover, MVR at ribbon synapses results in

[78,85]. It is therefore important to note that although the release of thousands of vesicles at single active zones

most central synapses have low Pr, mechanisms that ele- over prolonged periods of stimulation. The synaptic ribbon

vate Pr (such as frequency facilitation, decreased presyn- therefore appears to be a highly specialized structure that

aptic inhibition, or presynaptically expressed LTP) may promotes continuous MVR at primary sensory synapses.

also increase the probability of MVR [11,14,15,34,80,81].

MVR at excitatory and inhibitory hippocampal synapses

Functions of MVR throughout the brain Although initial studies rejected the MVR hypothesis

High-ﬁdelity transmission and adaptation at sensory [104,105], considerable variability in postsynaptic cur-

synapses: MVR par excellence at synaptic ribbons rents, as well as in anatomical and molecular properties,

Sensory processing requires neuronal circuits to transform led to the speculation that hippocampal synapses use MVR

graded information into a temporal code of action poten- to increase their dynamic range [11,30,83,106] (Box 1 and

tials. To continuously perform these computations synap- Figure 2). Indeed, modeling of SC–CA1 EPSCs also favors

ses need to adapt to sustained levels of transmitter release. the idea that MVR underlies current amplitude ﬂuctua-

Unlike conventional synapses that require action poten- tions [29,107]. A pioneering study in cultured hippocampal

tials to release vesicles, the presynaptic cells at many neurons identiﬁed MVR based on observations that multi-

primary sensory synapses typically operate via graded ple quanta interact with a common population of postsyn-

membrane potential changes that can last for several aptic receptors [12]. Consistent with this notion, NMDAR-

seconds. Here, vesicles are often tethered to a specialized mediated calcium inﬂux at single SC–CA1 spines increases

protein structure called a ribbon that is thought to main- in a stepwise manner during repetitive stimulation, dem-

tain and synchronize the release of hundreds of vesicles per onstrating that MVR underlies enhanced synaptic potency

second ([16,86–89], but see [90]). For example, at retinal during facilitation [11,15,38]. In addition, evidence from

bipolar cell terminals a 200 ms depolarization can release electrophysiological studies suggests that cleft glutamate

6000 synaptic vesicles at 50 synaptic ribbon release sites, concentration increases during trains of stimuli [17]. Final-

with each ribbon releasing 120 vesicles, a number similar ly, presynaptic modulators can control MVR in an input-

to the total number of docked vesicles at the ribbon plus and state-speciﬁc manner. For example, activation of the

those tethered to the ribbon [91]. In fact, vesicle pool presynaptic estrogen receptor b (ERb) induces acute po-

depletion is thought to be the major mechanism for spike tentiation of MVR speciﬁcally at low Pr SC–CA1 contacts

adaptation during sensory stimuli [92,93]. Release can [108]. Moreover, the transition from UVR to MVR may

occur with rapid and slow kinetics at ribbon synapses, underlie the expression of late LTP at CA3–CA1 synapses

resulting in multiphasic and/or large-amplitude excitatory [109]. Together these studies converge on the view that

postsynaptic currents (EPSCs) [89,94,95]. Several hypoth- MVR dynamically promotes reliable transmission at typi-

eses have been proposed to explain the near-synchronous cally low Pr hippocampal synapses and contributes to

fusion of several tens of vesicles, including coordination of activity- and context-dependent plasticity.

release sites and tight coupling of calcium channels to the

release machinery [21,89,96], and fusion of several vesicles Increasing reliability of cortical and subcortical synapses

to each other before exocytosis (compound fusion [97]). Heterogeneity of release mechanisms prevails in the

Recently, however, fusion pore regulation of UVR events cerebral cortex. Studies using statistical arguments and

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Feature Review Trends in Neurosciences July 2015, Vol. 38, No. 7

Box 1. Detecting multivesicular release

Variance-mean analysis (VMA) determines the quantal parameters q, Pr, non-equilibrium interaction of neurotransmitter and antagonist with

and N by relating synaptic current fluctuations (expressed as CV) to receptor binding sites [105]. Unlike a slowly unbinding, high-affinity

mean synaptic responses under various Pr conditions [143,144] antagonist that inhibits the PSC in an antagonist concentration-de-

(Figure 2Aiii). Together with anatomical reconstruction (Figure 2Ai,ii) pendent manner (Figure 2Bi), the extent of inhibition by the fast-off

and Gaussian histograms of synaptic response amplitudes, VMA serves antagonist also depends on transmitter concentration if the antagonist

to identify release modality [119,145–149]. Narrow-amplitude histo- dissociation rate is shorter than the presence of synaptically released

grams with equally spaced peaks are thought to reflect release of several transmitter. As a result, both transmitter and antagonist compete for

vesicles. Uniquantal models assume that peaks represent the number of receptor binding sites until transmitter is cleared [12,154] (Figure 2Bii).

release sites, each releasing at most one vesicle, whereas multiquantal This allows the fast-off antagonist to distinguish between MVR and

models assume that peaks reflect the release of multiple vesicles from a UVR because MVR results in higher transmitter concentrations in the

common active zone [11,20]. Single-peak histograms are thought to synaptic cleft than UVR (Figure 2Biii). The advantages of the fast-off

correspond to single-site UVR contacts [4,150], where synaptic varia- antagonist are easy implementation, fewer assumptions about quantal

bility (e.g., electronic distance from recording site, vesicle size, trans- parameters, the number of release sites and synaptic inputs. However,

mitter content, and receptor density) generate amplitude fluctuations it is unsuitable to report release heterogeneity between release sites,

([10,26,151], but see [28,29,40,152]). However, amplitude fluctuations and performs less reliably at closely spaced sites because spillover and

could also be consistent with saturation, spillover, or desensitization MVR might become indistinguishable [155].

during MVR. Optical quantal analysis monitors NMDAR-mediated calcium fluxes

MVR can be measured directly when the presynaptic neuron only in response to vesicle fusion at individual presynaptic boutons, and

forms a single contact with the postsynaptic neuron [13,127]. Here, requires the loading of the neuron with a calcium-sensitive fluorescent

spontaneous PSCs in have a significant fraction of double-peaked dye [15,18]. Similar to quantal current, the fusion of a vesicle causes a

events that occur within short time intervals, and statistical analysis quantal fluorescence change (Figure 2Ci). Hence, a stepwise increase

suggests that double peaks appear more often than expected by of fluorescence signals reflects an increase in the number of fusing

chance. Therefore, double peaks likely emerge from multiple vesicles vesicles, or MVR (Figure 2Cii,iii). Another optical technique uses the

released from a common active zone that interact with the same pH-sensitive green fluorescent protein pHluorin coupled to a vesicular

postsynaptic receptor population. transporter (e.g., vGlut–pHluorin) to monitor vesicle fusion at single

To detect MVR across a population of synapses, a receptor antago- sites and takes advantage of a pH-dependent increase in fluorescence

nist with a fast unbinding rate (typically a ‘low-affinity antagonist’) that occurs when the acidified vesicle is exposed to the basic extra-

[153] is a powerful but simple tool that takes advantage of the cellular milieu [38].

anatomical reconstructions ﬁnd evidence for UVR at intra- MVR recruiting local voltage-gated conductances or

cortical excitatory synapses [6,110,111]. Nevertheless, NMDARs, and shows that MVR can control dendritic

MVR can also enable faithful transmission at intracortical nonlinearities. Similarly to mAChRs, presynaptic GABAB

synapses in layer 4 (L4) of the somatosensory cortex. Here receptors at cortical pyramidal neurons reduce synaptic

L4 neuron terminals display high Pr and postsynaptic potency by decreasing Pr and MVR [115]. Therefore, the

receptor saturation that reliably initiate action potentials, modulation of the number of vesicles released at single

although MVR is less prevalent at L4 connections in the synaptic sites appears to control local signal integration in

visual cortex [19]. It is possible that the differential ex- addition to synaptic strength.

pression of MVR represents distinct coding of information

depending on content or valence. Dynamics of transmitter release in the cerebellum

Thalamic input to the cortex recruits robust feed-for- MVR has been studied in detail at cerebellar CF–PC

ward inhibition crucial for limiting cortical excitation and synapses. Following an action potential the near-synchro-

reﬁning sensory processing [112,113]. High Pr thalamocor- nous fusion of 3–5 vesicles per release site gives rise to

tical synapses onto interneurons display signiﬁcant MVR cleft glutamate concentrations of >10 mM that fully occupy

(up to seven vesicles), driving reliable inhibition onto AMPARs and generate large EPSCs [14,33] that result in

cortical pyramidal neurons [114]. However, the close spac- bursts of action potentials termed complex spikes [116–

ing of release sites hampers a clear distinction between 118]. Characteristic of high Pr, repetitive stimuli deplete

MVR and spillover at these contacts. In addition, corti- synaptic vesicles and cause a decrease in CF EPSC ampli-

cothalamic projections from L6 pyramidal neurons show tude [70,119]. Nonetheless, MVR and saturation are

use-dependent enhancement of synaptic strength mediat- thought to help speed recovery from depression, allowing

ed by increased Pr, MVR and, consequently, receptor satu- reliable transmission even during sustained CF activity at

ration [81]. In conclusion, while intracortical processing physiological frequencies observed in vivo (<30% depres-

seems to rely primarily on UVR synapses, the evidence sion at 2 Hz [39,120,121]). Firing at these frequencies also

suggests that MVR is important at the input and output causes a slowing of the EPSC waveform that is consistent

stages where signal transmission requires greater reliabil- with desynchronization of MVR within a release site,

ity. termed intra-site asynchrony [39]. Synaptic depression

In the striatum, cholinergic modulation of MVR and low and desynchronization of MVR generate complex spikes

levels of receptor saturation enhance the dynamic range of that propagate more successfully along the PC axon than

excitatory inputs onto medium spiny neurons. MVR is during near-synchronous MVR, thereby enhancing infor-

prevalent under basal conditions, but acetylcholine release mation transfer to PC target neurons. Downregulation of

from interneurons dramatically decreases synaptic poten- MVR by various presynaptic receptors (including GABAB,

cy (and MVR) by activating presynaptic muscarinic acetyl- adenosine, adrenergic, and metabotropic glutamate recep-

choline receptors (mAChRs) [18]. The decrease in MVR tors) [39,122,123] can disrupt associative plasticity of par-

accelerates the time course of excitatory postsynaptic allel ﬁber (PF) inputs [123], suggesting that modulation of

potentials (EPSPs), an effect that can be attributed to MVR at CF synapses has a role in cerebellar learning.

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Feature Review Trends in Neurosciences July 2015, Vol. 38, No. 7 (Ai) (Aiii)

75 mV 80 pA 10 ms )

2 4000

(Aii) 1 2 2000 N F = 26

Variance (pA Variance q = 24.1 pA

0 0 250 500

Mean current (pA) (Bi) (Bii) (Biii)

High P Control r Slow Fast 500 pA 10 ms

P Low r

200 pA

10 ms (Ci) (Cii) (Ciii)

1 µm G/R G/R G/R 0.5 0.2 0.1 50 ms 50 ms 50 ms

TRENDS in Neurosciences

Figure 2. Detecting multivesicular release (MVR). (Ai) Reconstruction from paired recordings of a presynaptic basket (soma and dendrites in black, axon in red) and

postsynaptic pyramidal cell (soma and dendrites in blue, axon in gray). Inset, electron microscopy (EM)-identified boutons shown at higher magnification. (Aii) Electron

micrographs show synaptic contacts between the presynaptic basket terminals and the pyramidal cell soma. Arrowheads indicate synaptic junctions. (Aiii) Presynaptic

action potentials (red) and resulting currents (blue) recorded under low release probability (Pr, left) and high Pr (right) conditions. Below, variance versus mean plot from

current responses recorded in (Aiii) was used to derive quantal parameters (bottom). Adapted from [30]. (Bi) (Top) Slow-off, high-affinity antagonist (labeled as ‘slow’)

occupies postsynaptic receptors (in orange). (Bottom) As a result of slow antagonist unbinding, synaptically released neurotransmitter (NT) binds only to the fraction of

receptors that is not bound by the antagonist. (Bii) (Top) Due to its fast off-rate a low-affinity antagonist (labeled as ‘fast’) rapidly unbinds from receptors. (Bottom)

Synaptically released transmitter competes with the antagonist for receptor binding sites. Therefore, inhibition by the antagonist depends on the transmitter concentration

and time course. (Biii) The slow high-affinity antagonist inhibits postsynaptic currents recorded under high and low Pr conditions to the same extent (left). The fast low-

affinity antagonist inhibits the excitatory postsynaptic current (EPSC) recorded under high Pr conditions to a lesser extent than at low Pr conditions (right). Adapted from

[39]. (Ci) Raster image of a dendrite with spines (red fluorescence) and the calcium transient after synaptic stimulation (green fluorescence). White lines and arrows indicate

the position of the line scan and the time of synaptic stimulation, respectively. Synaptic stimulation results in a rapid increase of the green fluorescence (calcium-sensitive)

that does not occur with the red fluorescence (calcium-insensitive). (Cii) The ratio of green/red fluorescence in response to a single (left) or a pair of synaptic stimuli (right)

shown by arrows. Failures of synaptic transmission can be clearly distinguished from successes. (Ciii) Average fluorescence responses to a single successful stimulus

(yellow) and to the second stimulus in a pair when the first stimulation produced a failure (green). The failure to both stimuli is also shown (black). Adapted from [15].

Despite the lack of direct synaptic connections between and provide the mechanistic details for the long-lasting

CFs and MLIs, recent studies indicate that glutamate modulation of PC ﬁring rate following CF activation in vivo

spillover from CFs evokes slow AMPAR and NMDAR- [124,125].

mediated EPSCs in MLIs that lead to a prolonged increase Desynchronization of MVR on a supra-millisecond time-

in ﬁring rate [62–64]. These ﬁndings suggest that MVR can scale is also evident at inhibitory synapses between MLIs

enhance the temporal and spatial spread of CF activation, [13] where 0–3 vesicles can be released per action potential

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Feature Review Trends in Neurosciences July 2015, Vol. 38, No. 7

at single release sites [126]. In these electrically-compact animals. The mechanism of enhancement is consistent

interneurons, desynchronized IPSCs occurring within time with an activity-dependent depression of NMDAR current

intervals of 5–20 ms could provide long-lasting inhibition that diminishes calcium inﬂux and the release of a tonic

that limits the integration of incoming PF-mediated exci- retrograde inhibitor from PNCs. Disinhibition of transmit-

tation. Consistent with MVR at MLI contacts, MLIs can ter release in turn increases MVR. Remarkably, both

also release multiple synchronized vesicles onto PCs via a behavioral sensitization to the stressor and enhanced

calcium store-dependent mechanism, resulting in minia- MVR last several days [20]. Therefore, potentiation of

ture IPSCs with exceptionally large amplitude (‘maxi- MVR may be the synaptic correlate of enhanced HPA axis

mini-IPSCs’) [37]. sensitivity.

Finally, at least a subset of low Pr PF terminals also Noradrenergic modulation of MVR may also regulate

undergoes MVR during long- and short-term activity-de- hormone secretion from magnocellular neurosecretory

pendent potentiation, albeit with considerable intersynap- cells (MNCs) in the hypothalamus. Noradrenaline aug-

tic heterogeneity [34,78,80,127,128]. Although the origin of ments presynaptic calcium via a1 receptor activation

this heterogeneity is unknown, differences in PF bouton and calcium release from ryanodine-sensitive stores, tran-

size, calcium dynamics, and sensitivity to G-protein-cou- siently enhancing synchronous MVR onto MNCs [140]. In

pled receptor activation have been discussed electrically-compact cells such as MNCs [141] large-ampli-

[129,130]. The high glutamate concentration generated tude MVR-unitary EPSCs are sufﬁcient to trigger action

by MVR is thought to activate extrasynaptic NMDARs potential ﬁring and, hence, release of vasopressin and

on MLIs [78], and extrasynaptic metabotropic receptors oxytocin, hormones crucial for the regulation of hydration,

on PCs and MLIs, as well as activating AMPARs on stress, hemorrhage, lactation, and parturition. Together,

Bergmann glia [33,52,131–133]. these results demonstrate that neuroendocrine output in

the hypothalamus partially underlies the short and long-

MVR during synapse maturation and development term regulation of MVR [142].

During postnatal development synapses undergo exten-

sive morphological and physiological changes. One promi- Concluding remarks

nent and well-studied example is the calyx of Held synapse We present abundant evidence that MVR is a common

of the auditory pathway [134]. Morphological reﬁnement mechanism for enhancing synaptic reliability and over-

(i.e., a transition from a cup-shaped structure to a more coming the stochastic variability of synaptic transmission.

fenestrated and claw-like morphology) occurs concomitant- Although the precise molecular mechanisms underlying

ly with a progressive speeding of the presynaptic action MVR remain unknown (Box 2), MVR determines synaptic

potential that decreases Pr and shortens synaptic delay behavior during repetitive and sustained activation,

[135]. MVR is less prominent at the mature compared to affects the temporal and spatial activation proﬁle of neu-

the immature calyx [136], allowing faster glutamate clear-

ance and less AMPAR desensitization and saturation Box 2. A mechanism for MVR

[66,136]. These coordinated changes enable this auditory

It is not known how MVR occurs. It appears most likely that many

synapse to ﬁre spikes at high frequencies (up to 1 KHz).

vesicles fuse independently with the plasma membrane (heterotypic

The expression of MVR at a given synapse may therefore be fusion) at most synapses [13–15,39]. However, alternative explana-

subject to developmental remodeling and track changes in tions have been considered. One example is the fusion of multiple

vesicles with each other before exocytosis (homotypic fusion or

Pr.

compound fusion). First observed in secretory cells [156], activity-

In the developing cerebellum, PCs receive multiple CF

and calcium-dependent compound fusion that could explain see-

inputs that undergo activity-dependent pruning. Strong

mingly coordinated MVR was also reported at ribbon synapses [97]

evidence suggests that the CFs with the greatest extent of and at the calyx of Held [157]. However, the conditions under which

MVR evoke the largest depolarization-dependent calcium compound fusion could occur at conventional synapses remain

unknown. Several lines of evidence argue against compound fusion

signals in PC dendrites, and consequently evade synapse

as a general mechanism for MVR: at the ribbon MVR can persist even

elimination [137,138]. The mechanisms that establish

in the presence of fast calcium buffers [21,158], MVR exhibits similar

MVR before pruning remain incompletely understood,

intra- and intersite temporal jitter [39], and proteins of the vesicle

but these results suggest that MVR may contribute to release machinery located on the plasma membrane and on synaptic

synapse reﬁnement. vesicles are asymmetrically distributed [159]. These observations

support the notion of multiple independent release events within a

release site [13–15,39]. In addition, fusion pore dynamics were hy-

Sensitization of homeostatic circuits

pothesized to govern peak transmitter concentration in the synaptic

Behavioral responses to external stimuli often intensify

cleft. Two fusion modes were proposed: incomplete fusion (‘kiss and

upon stimulus re-occurrence. Several lines of evidence run’), that frees transmitter slowly and might prolong the transmitter

suggest that MVR may be a synaptic correlate of such transient, or rapid full fusion, resulting in high transmitter concen-

tration [160,161]. However, the lack of kinetic changes in miniature

behavioral sensitization. One example is the stress re-

mEPSC (mEPSC) time course at varying Pr argues against such a

sponse of the hypothalamus–pituitary–adrenal (HPA) axis

mechanism to explain fluctuations in transmitter concentration

that generates a long-lasting sensitivity to repetitive [17,39]. In conclusion, the most likely factors in regulating the num-

stressors [139] that involves enhanced activity of parvo- ber of releasable vesicles, setting Pr, and determining the temporal

pattern of fusion that should be investigated in the context of MVR

cellular neurosecretory cells (PNCs) in the paraventricular

are density, recruitment, and type of presynaptic calcium channels,

nucleus of the hypothalamus. A recent study found poten-

calcium-buffering proteins, as well as proteins of the presynaptic

tiated excitatory synaptic transmission onto PNCs in ani-

vesicle-release machinery [84,98,162–165].

mals pre-exposed to a single stressor compared to naive

434

Feature Review Trends in Neurosciences July 2015, Vol. 38, No. 7

11 Conti, R. and Lisman, J. (2003) The high variance of AMPA receptor-

Box 3. Outstanding questions

and NMDA receptor-mediated responses at single hippocampal

synapses: evidence for multiquantal release. Proc. Natl. Acad. Sci.

The molecular determinants of UVR and MVR are unknown; the

U.S.A. 100, 4885–4890

composition of active zones that support MVR need further eluci-

12 Tong, G. and Jahr, C.E. (1994) Multivesicular release from excitatory

dation. Important factors may include calcium channel density,

synapses of cultured hippocampal neurons. Neuron 12, 51–59

subtypes and adapter proteins, calcium sensors of the release

13 Auger, C. et al. (1998) Multivesicular release at single functional

machinery, calcium buffering, availability of release sites, and

synaptic sites in cerebellar stellate and basket cells. J. Neurosci.

trans-synaptically acting proteins.

18, 4532–4547

The detailed mechanisms that govern the temporal pattern of MVR

14 Wadiche, J.I. and Jahr, C.E. (2001) Multivesicular release at climbing

(synchronized vs desynchronized) are unresolved.

ﬁber–Purkinje cell synapses. Neuron 32, 301–313

Presynaptic signaling pathways that underlie activity-dependent

15 Oertner, T.G. et al. (2002) Facilitation at single synapses probed with

regulation of MVR, especially during intermediate and short-term

optical quantal analysis. Nat. Neurosci. 5, 657–664

plasticity, need additional exploration.

16 Singer, J.H. et al. (2004) Coordinated multivesicular release at a

The consequences of MVR versus UVR for synaptic integration at

mammalian ribbon synapse. Nat. Neurosci. 7, 826–833

single spines and dendritic branches are not well studied. This

17 Christie, J.M. and Jahr, C.E. (2006) Multivesicular release at Schaffer

includes understanding how MVR contributes to the duration of

collateral–CA1 hippocampal synapses. J. Neurosci. 26, 210–216

the postsynaptic potential and to the recruitment of voltage-gated

18 Higley, M.J. et al. (2009) Cholinergic modulation of multivesicular

and calcium-dependent conductances.

release regulates striatal synaptic potency and integration. Nat.

Consequences of MVR for network function (spillover-mediated

Neurosci.

recruitment of inhibition, action potential propagation) have been

19 Huang, C.H. et al. (2010) Multivesicular release differentiates the

described in detail in the cerebellum. Whether similar phenomena

reliability of synaptic transmission between the visual cortex and the

apply to other circuits is unknown.

somatosensory cortex. J. Neurosci. 30, 11994–12004

20 Kuzmiski, J.B. et al. (2010) Stress-induced priming of glutamate

synapses unmasks associative short-term plasticity. Nat. Neurosci.

rotransmitter receptors and voltage-gated conductances, 13, 1257–1264

21 Graydon, C.W. et al. (2011) Sharp Ca nanodomains beneath the

and therefore controls synaptic integration, downstream

ribbon promote highly synchronous multivesicular release at hair cell

intracellular signaling, and the induction of plasticity. The

synapses. J. Neurosci. 31, 16637–16650

numerous examples of MVR discussed here illustrate that

22 Abenavoli, A. et al. (2002) Multimodal quantal release at individual

synapses differentially exploit MVR depending on their hippocampal synapses: evidence for no lateral inhibition. J. Neurosci.

functional demands. Nevertheless, several outstanding 22, 6336–6346

23 Watanabe, S. et al. (2013) Ultrafast endocytosis at mouse

questions remain (Box 3). In conclusion, we suggest that

hippocampal synapses. Nature 504, 242–247

MVR is a fundamental and simple mechanism for increas-

24 Tsodyks, M.V. and Markram, H. (1997) The neural code between

ing the dynamic range of synaptic transmission, and we

neocortical pyramidal neurons depends on neurotransmitter release

challenge the long-held hypothesis that MVR is a rare probability. Proc. Natl. Acad. Sci. U.S.A. 94, 719–723

phenomenon manifested exclusively at high Pr contacts. 25 Abbott, L.F. and Regehr, W.G. (2004) Synaptic computation. Nature

431, 796–803

26 Bekkers, J.M. et al. (1990) Origin of variability in quantal size in

Acknowledgments

cultured hippocampal neurons and hippocampal slices. Proc. Natl.

This work was supported by National Institutes of Health grants to J.I.W.

Acad. Sci. U.S.A. 87, 5359–5362

(R01NS065920) and H.v.G. (R01DC012938 and R01DC04274). We thank

27 Frerking, M. et al. (1995) Variation in GABA mini amplitude is the

Drs Monica Thanawala, Pascal Kaeser, Jessica Hauser, Anastasios

consequence of variation in transmitter concentration. Neuron 15,

Tzingounis, Jada Vaden, Linda Overstreet-Wadiche, and members of 885–895

the laboratory of J.I.W. for reading the manuscript.

28 Ishikawa, T. et al. (2002) A single packet of transmitter does not

saturate postsynaptic glutamate receptors. Neuron 34, 613–621

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